The vitamin K oxidoreductase (VKORC1) is required for the function of vitamin K-dependent (VKD) proteins, as it supplies the reduced vitamin K used in the carboxylation and consequent activation of these proteins. VKORC1 has broad biological impact, as VKD proteins function in hemostasis, calcium homeostasis, apoptosis, growth control, and signal transduction. VKORC1 is the target of the drug warfarin used to control hemostasis and mutations in VKORC1 cause severe bleeding, indicating the critical role of this enzyme in hemostasis. Understanding VKORC1 mechanism is therefore important, but at present is poorly defined. VKORC1 is inactivated during vitamin K reduction and requires reactivation by a redox protein. We developed an innovative approach for studying VKORC1, which led to the identification of an electron relay pathway in VKORC1 activation. VKORC1 is an integral membrane protein, and we showed that VKORC1 residues in a loop that resides outside of the membrane transfer electrons from a redox protein to membrane-embedded residues that reduce vitamin K. Revealing the importance of the extramembrane loop is significant, because the loop contains a large number of residues whose mutations cause warfarin resistance. We showed that VKORC1 has a dimeric structure, so these mutations likely exist as a heterodimer with wild type VKORC1 in warfarin resistant patients. We found that the dimeric structure allows VKORC1 to reduce vitamin K epoxide to quinone and then to the hydroquinone form used in VKD protein carboxylation. Interestingly, we found that warfarin resistant mutations are impaired in supporting VKD protein carboxylation because they cannot perform both reaction steps efficiently. Thus, warfarin inhibition is more complex than previously appreciated. Aim 1 proposes an innovative hypothesis that residues associated with warfarin resistance normally facilitate activity, and that mutations alter functions that may indirectly blok warfarin access. We will test this hypothesis by determining the activity and warfarin sensitivity of homo- and heterodimers of VKORC1. Aim 2 will generate a mouse model of warfarin resistance and test the hypothesis that a warfarin resistant mutant lowers VKD protein carboxylation in vivo, even in the absence of warfarin. Aim 3 will identify the redox protein that activates VKORC1. The proposed studies will be significant for understanding warfarin therapy and improving the production of VKD proteins for clinical applications.